Fermented Milk Products
A pleasant taste and easy digestibility are common characteristics of all kinds of fermented milk products. These products are typically nutritious and some have been shown to have medicinal benefit in treating and preventing infectious and other diseases. Fermented milk products can vary not only by the milk, but also by the method of preparation, organoleptic properties, chemical composition and content of microflora.
So-called lactic acid bacteria alone or in symbioses (species or strains of species) and often with yeasts can induce lactic acid fermentation. Lactobacillus acidophilus strains are preferred for generating fermented milk products, since these strains are known to remain in the large intestine of humans where they produce anti-microbial substances. Fermented milk products prepared using Lactobacillus acidophilus strains are nutritious as well as therapeutic. In addition to anti-microbial benefits, some milk products have been reported to stimulate gastric secretion and motor-secretory function of the entire gastrointestinal tract, reduce serum cholesterol and treat and prevent diarrhea.
Typically, Lactobacillus acidophilus fermented milk products include about 107-108 bacteria per 1.0 g of product and comprise about 2.8 wt. % of protein, 3.5 wt. % of fats, 88.5 wt. % of water, 1173 mg of essential amino acids in 1.0 liter (at a scaling factor of 6.38) [namely valine (157), isoleucine (156), leucine (267), lysine (215), methionine (71), threonine (126), tryptophan (41), phenylalanine (140)], and non-essential amino acids [namely alanine (80), arginine (100), aspartic acid (179), histidine (74), glycine (38), glutamic acid (492), proline (248), serine (153), tyrosine (151), cystine (20)].
A fermented milk product produced by sterilizing milk at 120xc2x0 C., cooling the milk to 30-34xc2x0 C. and then introducing from about 0.2 to 0.4 wt. % (milk) of Lactobacillus acidophilus strains NK1, KEW24 and 100AW taken in a 2:2:1 ratio, respectively, has been described (Russian Federation Patent SU, A, 1004471). Culturing was carried out at 30-34xc2x0 C. for 18-24 hours to ferment the milk up to a 180-210xc2x0 Th acidity (i.e. 1.62-1.89% lactic acid). The fermented milk produced was characterized as having an antagonistic activity against Shigellae sonnei, 99.9%; Shigella flexneri, 100%; Salmonellae, 99.3%; Proteus sp., 99.9%; and possessing a proteolytic activity of 62.5%. The product was found to be resistant to antibiotics (e.g. monocyclin, neomycin, kanamycin, polymixin, penicillin, erythromycin, streptomycin and tetracyclin). Moreover, the product was reported to possess a resistance to drying over the range of 45-58% viable cells on freeze-drying, a storage stability over the range of 80-90% viable cellsxe2x80x94(in the dry form) at room temperature. However, the product has shown no benefits in treating gastrointestinal disease, premature infants or other conditions.
Fermented milk products have also been made by culturing the L.acidophilus K1, K5 or K10 strains (107 to 108 bacteria per gram), which are phenol-resistant, possess an increased antibiotic activity with respect to bacteria of the Escherichia coli group including pathogenic microorganisms, with cow milk, seed oil and refined sugar. Their beneficial effect in the digestive tract of infants has been described. (xe2x80x9cProducing Child""s Milk Productsxe2x80x9d, Moscow, Light and Food Industry, 1982, p. 67.)
A fermented milk product prepared with these strains has been reported to contain the following: protein, 1.8-1.9 wt. %; fat, 3.5 wt. %; carbohydrates, 7.0 wt. %; mineral substances, 0.5 wt. %; water, 87.1-87.2 wt. %. The content (wt. %) of essential amino acids in the fermented milk protein, which result from partial proteolysis of proteins during culturing, was reported to be as follows: lysine, 7.0; threonine, 2.6; methionine, 09; valine, 5.5; phenylalanine, 4.4; leucine, 14.8; isoleucine, 3.2; histidine, 2.5; tryptophan, 2.1. The product is further reported to have an acidity of about 50-70xc2x0 Th (from 107-108) and an energy value of about 65 kilocalories per 100 grams of product (xe2x80x9cUsing Acidophilic Mixtures for Feeding Healthy and Sick Infants of the 1st Year of Lifexe2x80x9d, Moscow, 1980, p. 4-7).
Although the fermented milk product contains protein, fatty acids, carbohydrates, vitamins and minerals, all of which positively influence intestinal biocenosis and can stimulate the immune system, the microorganisms do not produce vitamins. The following artificially supplied vitamins comprise the fermented milk product described above: (vitamins, wt. %) A-0.06; D2-0.01; E-10.0; C-5.0; B10.05; B20.07; B6-0.04 ; PP (biotin)-0.4.
Lactobacillus acidophilus 
Lactobacillus acidophilus are microaerobic, Gram-positive, homofermentative, and immobile. They are granular and non-granular asporogenic bacteria of 2-10xc3x970.7-0.8 xcexcm in size. Cells are disposed singly, in short or long chains, and are characterized in that they do not reduce indole and do not form skatole nor dilute gelatine. In addition, L. acidophilus does not possess a hemolytic activity. However the bacteria do ferment the following: lactose, glucose, sucrose, fructose, mannose, galactose, maltose, raffinose, starch, dextrin, sorbitol, mannitol, dulcitol, but not inositol on selective agar nutrient media in Burri tubes or Petri dishes. They form subsurface colonies which are shallow, rarely smooth (S-forms), most commonly in the form of a ball of entangled threads (R-forms). The minimal growth temperature is at least 20xc2x0 C. and preferably 35-39xc2x0 C. Temperatures of above about 68-70xc2x0 C. are typically lethal. When cultured in milk, L. acidophilus strains result in a maximum acidification of about 300-360xc2x0 Th, a phenol resistance of about 0.9-1.0%, a phthalaso-resistance of about 0.9-1.0%, and a synthomycin resistance of about 0.003%.
A. Er-2 strains
Some L. acidophilus bacteria, the Er-2 group, are known flavin producers. Examples of Er-2 strains include Nos.: 317/381, 317/393, 317/401 or 317/389 (L. A. Erzinkyan; Biological Features of Some Lactic Acid Bacteria Strains, Academy of Armenian Sciences Publishers, Yerevan, 1971, pp. 79-96, 172). In addition, Er-2 strains of Lactobacillus acidophilus are known to produce a substantial amount of antimicrobial substances which are beneficial for humans. These substances have been shown to suppress growth and development of Gram-positive (e.g. Staphylococcus aureus) and Gram-negative (e.g. Escherichia coli) bacteria including, without exception, all dysentery and dysentery-like diseases induced by bacteria.
B. Lactobacillus acidophilus N.V. 317/402
Fermented milk products, which possess high dietary versatility, easy assimilability, curative properties, and a high nutritive value have been obtained using Lactobacillus acidophilus strain N.V. Er 317/402 (Nauka Industry Co. Ltd., Japan). When preparing a fermented milk product, 1.0 wt. % of this strain is introduced into whole milk at 28-40 C., wherein milk clotting occurs within 5-8 hours ( Russian Federation Patent No. SU, A, 1635573). The strain used possesses a slow acid-forming capability. A maximum acidification value reaches about 360xc2x0 Th.
Bacteria of this strain are capable of producing a substantial amount of antibiotics, which are harmless and in fact beneficial for humans (particularly infants). These antibiotics suppress growth and development of both Gram-positive and Gram-negative bacteria including all bacterial inducers of active forms of gastrointestinal diseases. The presence of the strains in vitro have resulted in growth suppression zones for the following organisms: Staphylococcus aureus (30-35 mm), Escherichia coli (20-25 mm), Salmonella typhimurium (20-23 mm) and Klebsiella (20-22 mm). Thirty-six years of investigations have shown absence of phagolysis ( i.e. destruction of leukocytes necessary in wound healing and inflammation control) with this strain.
Lactobacillus acidophilus N.V. 317/402 was produced in 1949 by a directed selection in Yerevan (The Armenian Republic). A stock culture was isolated from the feces of healthy newborns. The bacteria can be inoculated into a liquid nutrient medium and stored at 5-8xc2x0 C. Alternatively, the culture can be lyophilized (freeze-dried) and stored at 5-8xc2x0 C. The strain can also be stored in liquid nitrogen at xe2x88x92193xc2x0 C. Lactobacillus acidophilus N.V. 317/402 are facultative anaerobes. To maintain the strain, the culture should be reinoculated once every 1-1.5 months. Milk is the preferred medium for propagating the culture.
The strain has been characterized as being homofermentative, immobile Gram-positive, microaerophilic, asporogenic bacilli of about 2-20xc3x970.8-0.9 xcexcm in size. Cells are disposed singly or in the form of short chains. Subsurface shallow colonies form on selective agar media, most commonly as balls or in the form of mixed up threads (R-forms). The strain does not reduce nitrates, form skatole or indole, dilute gelatine, nor possess a hemolytic activity. It does ferment the following: lactose, glucose, sucrose, fructose, mannose, galactose, maltose, raffinose, starch, dextrin, sorbitol, mannitol, and dulcitol, but does not ferment inositol. In milk the strain results in a phenol resistance of about 0.9-1.0% and a synthomycin resistance of about 0.003%.
Lactobacillus salivarius 
Bacteria of this species are Gram-positive, homofermentative, immobile, granular and non-granular asporogenic. Cells are disposed singly, in short or long chains, they do not reduce nitrates and do not form indole or skatole, nor dilute gelatine. The bacteria do not possess hemolytic activity. They do ferment the following: galactose, lactose, maltose, mannitol, sucrose, esculin, melibiose, raffinose, rhamnose, sorbitol. A limiting acid formation is 230-260xc2x0 T. They do not form gas from glucose, and do not grow at 15xc2x0 C. An optimal culturing temperature is +39 or +40xc2x0 C. Lactobacillus salivarius subsp. salicinius strain 11742 and Lactobacillius salivarius subsp. salivarius strain 11741 are available from the American Type Culture Collection.
In one aspect, the invention features a bacterial cultures or ferments that can be cultured in milk. In one embodiment, the ferment comprises Lactobacillus acidophilus strain N.V. Er 317/402 and at least one strain from the group Er-2, preferably in a ratio of 1-4:1, respectively. In another embodiment, the ferment comprises Lactobacillus acidophilus strain N.V. Er 317/402 and at least one strain from the group Er-2, preferably in a ratio of 1-4:1, respectively and further comprises a Lactobacillus salivarius in a 1:1 ratio with the total Lactobacillus acidophilus content.
In another aspect, the invention features fermented milk products produced by culturing milk with an appropriate amount of the novel ferments. In one embodiment, the fermented milk product is a liquid (e.g. a fermented milk drink or infant formula). In another embodiment, the milk product is solidified (e.g. into a yogurt, curd or ice cream-like composition). In a further embodiment, the fermented milk product is a powder, that can be reconstituted, for example, into a liquid or solidified milk product or prepared into a pharmaceutical composition (e.g. an orally administrable capsule, suppository or topical ointment).
The above and other features of the invention will now be more particularly described and pointed out in the following Detailed Description and Claims.
The invention features biologically active fermented milk products prepared using novel cultures of lactic acid bacteria of the species Lactobacillus acidophilus. The resulting products possess nutritive, prophylactic and therapeutic properties.
The novel culture or ferment comprises a combination a known Lactobacillus acidophilus strain of the group Er-2 and the known strain Lactobacillus acidophilus N.V. Er 317/402. Example of strains of the Er-2 group of Lactobacillus acidophilus include:
317/381, 317/393, 317/401 and 317/389. A preferred ferment is comprised of at least one strain from the Er-2 group of Lactobacillus taken in a ratio of 1-4:1 with the N.V. Er 317/402 strain of Lactobacillus acidophilus. 
The antibiotic activity of the culture, particularly against organisms found in the intestines of humans and non-human animals, can be further increased by combining a Lactobacillus salivarius strain with the novel culture described above. Preferred Lactobacillus salivarius strains include strain numbers 1588, 11741, 11742 and 29602. Preferably the Lactobacillus salivarius strain is in a 1:1 ratio with the total content of Lactobacillus acidophilus. 
Fermented milk products produced in accordance with the present invention, may be prepared from virtually any milk obtained from any source (e.g., cow, goat, sheep, mare, buffalo, human etc.) and of any composition (e.g whole, low-fat, fat-free, cream or otherwise modified). In addition, a variety of xe2x80x9cadditivesxe2x80x9d can be included either before, after or while the milk is being cultured. Examples include color (e.g., beta-carotene, anatto, tumeric, paprika and FD and C dyes); flavors, aromas or sweeteners (e.g., fructose, sucrose, glucose, mannose, levulose, galactose, maltose, raffinose, melibiose, starch, dextrin, sorbitol, mannitol, dulcitol, inositol, aspartame, honey, vanillin, chocolate, orange, cherry, peach, strawberry, pineapple, raspberry, guava, mango, banana, blueberry; emulsifiers and/or thickening agents (e.g., phosphatides such as egg yolk, soybean or corn lecithin or substances such as mono- or di- glycerides, gum arabic, tragacanth, arabinogalactan, carrageenan, furcellaran, sodium alginate, bean gum, gum xanthan, guar gum, and apple pectin); preservatives, vitamins and antioxidants (e.g., vitamins A, C, D, E, B-1, B-5, B-6, zinc, selenium, calcium, alpha-tocopherol, glutathione, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and cysteine).
Preferably the fermented milk products are prepared by introducing an appropriate amount of the novel culture into pasteurized milk and allowing fermentation to proceed at an appropriate temperature and for an appropriate period of time to obtain a product of suitable acidity. Preferably, the acidity of a food product is in the range of about 60-165xc2x0 Th (0.5400-1.4850% lactic acid). However for use as a pharmaceutical, as described below, the fermented milk product can have an acidity up to about 270-280xc2x0 Th. 280xc2x0 Th (0.5400-2.2600% lactic acid) and contains about 106-109 culture bacteria per gram of product. An appropriate fermenting temperature is a temperature that accomodates the metabolism of the culture organisms. Optimal temperatures for culturing Lactobacillus acidophilus strains range from about 20xc2x0 to about 50xc2x0 C.
If the milk to be fermented has not been pasteurized, sterilization can be accomplished using methods which are well-known in the art. For example, the milk can be heat-treated at an appropriate temperature, which can vary according to the type of milk, the ultimate manufactured product and the needs of the consumer. Many years of monitoring the production of the product described herein by constant quality indices, (e.g., acidity, amino acid composition and vitamin content) have shown consistent and stable manufacture and storage.
The resulting fermented milk products are highly nutritious. The products have typical energy values of about 20 to about 540 kilocalories (per 100 gms of product). In addition, they are typically comprised of the following (wt. %): proteins, 0.35-34.0; fats, 0.05-36.0; carbohydrates, 1.5-70.0; organic acids (in terms of lactic acid), 0.6-56.0 wt.; vitamins (in 100 grams), 0.95-15.9 mg; water, 3.7-94.6 wt. The protein component of the product is typically comprised of the following essential amino acids (in percent based on the sum of amino acids in the product): valine, (10.4-10.77); isoleucine, (9.6-9.7); leucine (13.8-14.1); lysine (3.6-5.0); methionine (2.7-3.1); threonine (0.9-1.1); tryptophan (0.5-0.7); phenylalanine (8.4-8.5).
Additionally, the fermented milk products typically contain the following vitamins (per 100 gms of product): Vitamin A, traces-0.05 mg; Pantothenic acid, 0.10-0.19 mg; Vitamin C, 0.5-0.90 mg; Biotin, 0.002 mg; Vitamin E (tocopherol), traces-0.2 mg; Nicotinic acid, 0.040-0.047 mg; Niacin, 0.09-0.14 mg. Vitamins of the group B, 0.218-0.315 mg, including: Vitamin B1 (thiamin), 0.04-0.07 mg; Vitamin B2 (riboflavin), 0.15-0.20 mg; Vitamin B6 (pyridoxine), 0.025-0.04 mg; folic acid, 0.003-0.005 mg. In fact, milk fermented with the novel cultures has been found to have enhanced vitamin content particularly with regard to group B vitamins, vitamin C, pantothenic acid, biotin, vitamin E, nicotinic acid, and niacin.
In addition to being nutritious, fermented milk products containing the novel cultures of microorganisms are active antagonists of opportunistic and pathogenic microorganisms and therefore their presence in vivo is beneficial for treating diseases or conditions that are caused by or contributed to by opportunistic or pathogenic microorganisms. Examples include gastrointestinal diseases such as dysbacteriosis, salmonellosis, constipation, colitis, lactose intolerance, dysentery and common diarrheas, (resulting for example from infection by microorganisms such as Escherichia coli, Enterobacter sp. Salmonella sp. and Proteus sp.), hemolytic jaundice (resulting for example from infections by microorganisms such as Staphylococcus aureus, Klebsiella sp. and Pseudomonas aeruginosa), omphalitis in newborns, in obstetric-and-gynecological practice (e.g. treating vulvovaginitis), other pyo-inflammatory diseases; for treating urethritis, bladder infections, allergies, sinusitis, rhinitis, high serum cholesterol, blood, liver, kidney diseases, atrophy and dystrophy with children, sepsis, certain cancers (e.g. colon, colo-rectal and breast), and infected wounds. As studies reported in the following Examples have shown, fermented milk products containing the claimed symbiotic microorganisms are inducers of xcex1- and xcex3-interferons and therefore enhancement of a subject""s (e.g. human""s or animal""s) immune response.
Since the cultures are well adapted to the gastrointestinal tract of humans and animals and eliminate disease causing organisms therein, administration of the cultures is particularly useful for treating or preventing intestinal dysbacteriosis (i.e. a change to the indigenous microbial flora in a human or animal resulting from the presence of antibiotics, chemotherapeutics or infectious organisms (e.g. Salmonella sp.)) Further, the cultures have been found to be resistant to high phenol concentrations, and certain toxic chemicals, chemotherapeuticals, and antibiotics and therefore would be expected to remain in a subject""s gastrointestinal tract for a period of time after initial introduction.
The fermented milk product can be prepared as a liquid (e.g. a fermented milk drink or infant formula). To prepare fermented milk products suited for feeding and treating premature and other children of all ages, the product resulting from culturing can be subjected to separation under aseptic conditions to give a liquid fraction of the following composition: proteins, 0.35-0.8 wt. %; fats, 0.05-0.20 wt. %; carbohydrates, 1.5-3.8 wt. %; organic acids (in terms of lactic acid), 0.70-3.15 wt. %; vitamins, 1.18-1.54 mg/100 g; water, 3.7-94.6 wt. %. The protein fraction remaining upon separation of the liquid fraction generally has the following composition: proteins, 5.5-11.0 wt. %; fats, 0.1-6.0 wt. %; carbohydrates, 1.6-2.0 wt. %; organic acids (in terms of lactic acid), 1.0-2.0 wt. %; vitamins, 1.2-1.56 mg/100 g; water, 83.4-86.6 wt. %. Alternatively, the milk product can be thickened into a semi-solid or solid composition (e.g. a yogurt, butter, cheese or ice cream-like composition).
Further, the fermented milk product can be prepared as a powder (e.g. by freeze drying). Experiments have shown that a powder prepared from a fermented milk product as disclosed herein can be stored for prolonged periods and can be readily reconstituted, for example, into a liquid or solidified milk product. A preferred fermented milk powder contains: proteins, 6.0-12.9 wt. %; fats, 0.9-3.3 wt. %; carbohydrates, 26.7-70.2 wt. %; organic acids (in terms of lactic acid), 15.4-56.0 wt. %; vitamins (per 100 grams of the product), 11.8-15.4 wt. % and water, 4.0 wt. %. A particularly preferred powder is obtained by freeze drying a fermented milk product, which has a 60-280xc2x0 Th (0.5400-0.5600 lactic acid) acidity to a water content of not more than 4 wt. %. The powder can be reconstituted to prepare a liquid (e.g. baby food formula or fermented milk drink) or thickened into a semi-solid or solid composition.
In view of their recognized medicinal value, the fermented milk powder or lyophilized culture can be formulated into pharmaceutical compositions (e.g. a capsule, suppository or ointment) for enteral or topical administration. In order to obtain a concentrated product suitable for medical use, in vitro and in vivo fermentation can be carried out up to an acidity of about 270-280xc2x0 Th.
A preferred pharmaceutical prepared from the liquid fraction obtained after fermentation and which has been separated under aseptic conditions is subjected to freeze drying up to a humidity of not more than about 4 wt. % to prepare a powdered substance comprising the following: proteins, 7.7-12.9 wt. %; fats, 0.8-3.3 wt. %; carbohydrates, 26.7-70.2 wt. %; organic acids (in terms of lactic acid), 15.4-56.0 wt. %; vitamins (in 100 grams), 11.80-15.4 mg; water, 4.0 wt. %. Storage of the product, even under elevated temperatures has been found not to alter the biochemical properties.
A pharmaceutical composition comprising the fermented milk product can be enterally administered to a subject (e.g human or animal) alone, or in conjunction with a pharmaceutically acceptable carrier. As used herein, the phrase xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is intended to include substances that can be coadministered with an appropriate fermented milk product and allows the invention to perform its intended function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the invention also falls within the scope of the present invention.
Alternatively, pharmaceutical compositions, which have been prepared as ointments, tinctures, creams, gels, solutions, lotions, sprays, suspensions and suppositories can be topically administered.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.